We found previously that C57BL/6J (B6) and C3H/HeJ (C3) mice are susceptible and resistant, respectively, to lung injury induced by exposure to hyperoxia (100% oxygen). Using BXH Recombinant Inbred (RI) strains and a B6C3F2 cohort, we completed genome-wide screens for chromosomal loci of susceptibility genes that control hyperoxia-induced pulmonary injury. The analyses identified significant and suggestive susceptibility quantitative trait loci (QTLs) on chromosomes 2 and 3, respectively. A candidate gene within the chromosome 2 QTL is Nfe2l2 (nuclear factor, erythroid derived 2, like 2 or Nrf2), which encodes a transcription factor NRF2 (NF-E2 related factor 2). NRF2 has been identified as an antioxidant response element (ARE)-mediated positive regulator of detoxifying enzyme genes for protecting cells against electrophile toxicity, oxidative stress, and carcinogenicity. To begin testing the hypothesis that Nrf2 is a candidate gene for differential susceptibility to oxygen toxicity in B6 and C3 mice, sequence analysis of Nrf2 from B6 and C3 mice was also done to identify the potential molecular basis. A potentially important variation was identified: B6 mice possess a T to C substitution at -336, which is predicted to add a Sp1 transcription factor-binding site in B6 mice compared to C3. The polymorphism segregated with SSLP genotypes (D2Mit248 and D2Mit94) at the peak of the chromosome 2 QTL, and the susceptibility phenotype in B6C3F2 mice. These data suggested that the polymorphism is potentially an important determinant of susceptibility to hyperoxic lung injury in this model. We are currently utilizing in vitro cell transfection studies to confirm the importance of the polymorphism. We further tested the hypothesis that NRF2 contributes to pulmonary protection against hyperoxic injury in mice by exposing mice with site-directed mutation (knockout) of Nrf2 (Nrf2-/-) and wt mice (Nrf2+/+) to hyperoxia. Pulmonary injury was significantly greater in Nrf2-/- mice compared to Nrf2+/+ mice after 48 and 72 hr hyperoxia exposure. Hyperoxia also markedly elevated expression of NRF2 mRNA and DNA-binding activity of NRF2 in lungs of Nrf2+/+ mice.